Adipose tissue is an essential component of our body, the excessive accumulation of adipose tissue will lead to obesity that predisposes to hypertension, diabetes, cardiovascular diseases and so on. In recent years, it is well established that adipose tissue is not only working as an energy storage depot, but also increasingly viewed as an active endocrine organ with a high metabolic activity secreting numerous adipocyte-derived hormones referred to as adipokines [1
]. These adipokines play a major role in regulating food intake, energy metabolism and inflammatory response acting on the brain, liver, muscle and other tissues via autocrine, paracrine and endocrine pathways [3
]. It is thought that the imbalance between the inflammatory adipokines leads to monocyte/macrophage infiltration and a vicious cycle which eventually results in systemic inflammation compromising cardiac, vascular and metabolic tissues [5
]. Hence, understanding the expression profile and mechanisms controlling of these inflammatory adipokines will provide potential therapeutic opportunities to tackle the global epidemic of metabolic disease.
The adipose tissue in mammals has been divided into two types. The white adipose tissue (WAT) stores excess energy in the form of triglycerides. Conversely, the brown adipose tissue (BAT) is specialized in energy expenditure and responsible for non-shivering thermogenesis called adaptive thermogenesis [7
]. What is more, recent studies have identified an intermediate type of adipocytes which exist within certain WATs in mice and rats [9
]. These cells are named beige or brite (brown in white) cells and they express the high level of uncoupling protein-1 (UCP-1) and mitochondria genes, and also show the multilocular lipid droplets like the morphological characteristic of BAT [10
]. These cells become more prominent upon prolonged stimulation by cold or β3
-adrenoceptor agonists such as CL316,243 [12
]. Compared to BAT, WAT is the major source of inflammatory adipokines [13
]. One of the most important inflammatory adipokines is the 16 kDa cytokine-like protein, Leptin, which is the product of the obese gene (ob) and has a variety of physiological effects [16
]. It helps to keep energy balance through the inhibition of food intake and the consumption of energy expenditure [4
]. Besides, Leptin also acts on multiple types of immune cells, such as neutrophils, monocytes/macrophages and T cells, to promote the release of inflammatory cytokines [18
Another important adipokine, Adiponectin (also known as AdipoQ), contributes to enhance insulin sensitivity in obese mouse [22
], normalize lipid metabolism dysfunction, inhibit energy expenditure and lead to weight loss in diet-induced insulin-resistance mice [23
]. Apart from its metabolic functions, Adiponectin is also implicated in the regulation of immune responses. Many lines of evidence show that Adiponectin has anti-inflammatory functions due to its modulation of macrophage phenotype [14
]. Additional adipokines include interleukin-10 (Il-10), Monocyte chemoattractant protein–1 (Mcp-1) and Tumor necrosis factor-α (Tnf-α). These adipokines are classified into anti-inflammatory and pro-inflammatory depending on their biological properties.
Since the previous studies of our group have indicated that cold-induced remodeling of WAT acquired some characteristics of BAT, including the thermogenic capacity and molecular morphology [25
], we propose that the expression profile of inflammatory adipokines in WAT will also be “browning” and get some features of BAT during the beige adipogenesis. Hence, the main objective of this study is to investigate the expression levels of main inflammatory adipokines in both BAT and WAT during cold exposure. The effect of browning by β3
-adrenoceptor agonist (CL316,243) on the expression of main inflammatory adipokines has also been assessed in primary mice brown adipocytes (BA) and white adipocytes (WA) in vitro
It is well known that cold exposure or β3
-adrenoceptor agonist would induce the browning of adipose tissues with the beige adipogenesis. However, the origin of beige cells within WAT and gene expression pattern was distinct from those of BAT [26
]. Recently, we reported that the time course effects of the cold-induced browning on adipose tissues exhibited depot-specificity [25
]. It remains poorly documented whether the expression profiles of inflammatory adipokines also alter differently during the browning process. In this study, we have addressed this by characterizing the temporal changes in expression of inflammatory adipokines induced by 1–5 days of cold exposure in mouse iBAT, sWAT and eWAT. In addition, the expressions of those in BA and WA in response to β3
-adrenoceptor agonist (CL316,243) have also been studied. We find that WAT exhibits a dominant source of inflammatory adipokines and plays a central role in the regulation of systemic metabolism under basal condition. While during the browning process, the expressions of inflammatory adipokines were dynamically changed both in vivo
and in vitro
, with depot-specificity among the adipose tissues.
As the biggest endocrine organ in the body, adipose tissues product and secrete various adipokines [15
]. Different kind of adipose tissue depots can be distinguished by their profile of secretion of adipokines [14
]. The current notion in the field is that WAT is the main source of inflammatory adipokines [31
]. Our studies support this view when the expression levels of inflammatory adipokines in sWAT and eWAT are compared with that of iBAT.
Leptin, primarily secreted by adipose tissues, plays an important role in regulating energy balance and body mass [32
]. Our present data suggested that Leptin
mRNA levels decreased significantly since the early stage of browning process, which was consistent with the previous studies [33
]. However, we observed the decreased plasma level of Leptin only at Day 5. This result was consistent with the previous study that the Leptin in serum did not alter upon the acute cold exposure [37
] but significantly decreased after the chronic cold acclimatization [38
]. Collectively, our data indicated the hysteresis quality of the alteration in the systemic level of Leptin.
Similar expression pattern was observed in Adiponectin. We have shown that the expression profile of Adiponectin
mRNA was decreased significantly in sWAT and iBAT, but up-regulated in eWAT. However, Adiponectin protein was increased in sWAT at Day 5 of cold exposure. This discordance between mRNA and protein levels of Adiponectin was possibly due to the low efficiency of protein biosynthesis in the cold environment in sWAT. Interestingly, in the early phase of browning process, there was a reversal of Adiponectin
gene expression in both white and brown adipocytes, which was up-regulated first and then declined significantly after 12 h treatment of CL316,243 in vitro
. There is also another report which claimed that chronic cold exposure induced Adiponectin expression in subcutaneous fat [39
]. The reasons for the discordant observations are more likely to be due to the different control groups of mice (30 °C versus
RT, which is below thermoneutrality for mice). However, it is noteworthy that the plasma levels of Adiponectin did not fluctuate during cold exposure. This was in line with the previous study which demonstrated a significant decrease in Adiponectin
mRNA in adipose tissues after cold exposure or β3
-agonists treatment but no alteration in serum Adiponectin level [40
]. These observations might be attributed to the following reasons. First, adipose tissue is both the primary site of Adiponectin synthesis and a major target organ for Adiponectin actions so that the main regulatory manner of Adiponectin is autocrine or paracrine, but not endocrine. Second, the expression of Adiponectin on plasma level might also exhibit hysteresis quality after the gene level. Taken together, these findings highlight a complicated role of Adiponectin response to cold or β3
-agonists, which needs to be further investigated in the future.
In addition to our recent report that there were regional differences in the fat mass, cellular morphology and browning markers among the adipose tissues in response to cold [25
], the present study showed the alteration of inflammatory adipokines expression during the browning was depot-specific either. Although the expression of Il-10
was unchanged in iBAT during the cold exposure, their expression in BA in vitro
was enhanced and then came down to the basal line within 24 h, indicating that the expression of some inflammatory adipokines was activated rapidly at the very early stage of the browning process. However, the inflammatory adipokines in sWAT showed different expression profile. Except for Tnf-α
, whose expression exhibited a rising trend both in vivo
and in vitro
, the expression level of inflammatory adipokines (especially the pro-inflammatory adipokines) was up-regulated within the first few hours and then decreased in the following days in sWAT, which showed a similar expression profile of inflammatory adipokines in iBAT. This might be attributed to the important role of sWAT as an endocrine organ which participates in the long-range general regulation of metabolism. It is tempting to speculate that the modulation of the endocrine function in sWAT was in favor of the inflammatory balance during the browning process. Interestingly, the expression of many inflammatory adipokines differs between sWAT and eWAT upon cold exposure. In spite of the down regulation of inflammatory adipokines in sWAT, the expressions of such adipokines in eWAT were obviously increased during the cold stimulation. This finding is not surprising given the well-documented different propensity to accumulate beige cells between sWAT and eWAT and the differences in adipocyte biology in rodents [25
]. It is also consistent with the thesis that WAT is regionally distinct in terms of function, adipokines production, and inflammation; even the density of solitary adipose tissue macrophages in sWAT is much lower than that in eWAT [43
]. Moreover, the decrease expression of inflammatory adipokines in sWAT may induce the compensatory increase of that in eWAT. As an important part of visceral fat, eWAT is thought to play an important role in the etiology of various metabolic disorders [44
]. Understanding the mechanisms of WAT/BAT phenotypic conversion in eWAT might point the way toward novel therapies for these diseases. Therefore, the role of eWAT in the modulation of inflammation under cold exposure still warrants further investigation.
All these present observations essentially accord with the findings that cold exposure helps to prevent obesity, insulin resistance and other metabolic disorders. It is based on the regulation in endocrine and metabolism during the cold-induced beige adipogenesis among the adipose tissues [26
]. While someone found that people living in northern hemisphere are fatter because of cold climate. We think this contradictory phenomenon may be due to the perennial rather than the acute cold acclimation, which still needs additional study.
4. Materials and Methods
4.1. Mouse Colonies and Cold Exposure
All male mice were in the C57BL/6J background, and obtained from the Medical Experimental Animal Center of Xi’an Jiaotong University (Xi’an, China) at 8 weeks of age. They were singly kept in a specific pathogen-free (SPF) environment with a 12:12-h light–dark cycle and had free access to water and standard chow diet. After one week of adaption, they were randomly divided into six groups: room temperature (RT) for 5 days, and cold exposure (Cold) for 1–5 days (n
= 6 for each group), as in our previous study [25
]. All animal studies were approved by the Ethical Committee of Xi’an Jiaotong University, China. This study was approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University (Number: XJTU-2012-03-06-0037). Upon completion of the experiment, the blood of mice was collected by retro-orbital bleed. The mice were subsequently sacrificed and the iBAT, sWAT and eWAT [42
] were removed and frozen in liquid nitrogen for RNA and protein extraction.
4.2. Adipocyte Culture
For the culture of primary mice BA and WA, iBAT and sWAT were isolated from 3-week-old C57BL/6J male mice. The white preadipocytes were maintained in DMEM/F12 (Gibco–BRL Laboratories, Grand Island, NY, USA) supplemented with 2 mM l-glutamine, 100 U/mL penicillin (Sigma, St. Louis, MO, USA) and 10% fetal bovine serum (FBS) (Gibco–BRL Laboratories), while the brown preadipocytes were maintained in DMEM high glucose medium (Gibco–BRL Laboratories) supplemented with 5 mM HEPES, 100 U/mL penicillin (Sigma) and 20% FBS (Gibco–BRL Laboratories) both at 37 °C and 5% CO2. To induce differentiation, postconfluent preadipocytes were cultured in a standard differentiation medium (the DMEM or DMEM/F12 contained 850 nM insulin and 1 nM triiodothyronine). For the first three days of differentiation period, 0.5 mM 3-isobutyl-1-methylxanthine, 0.125 mM indomethacin, 1 µM dexamethasone and 1 µM Rosiglitazone were also added. The cells were fully differentiated after 9 days of culture in the differentiation medium.
The cells were treated with CL316,243 (2 µM, Tocris Bioscience, Bristol, UK) for 1, 2, 3, 4, 5, 6, 12 and 24 h after the deprivation of FBS for 12 h. After CL316,243 treatment, mature adipocytes were lysed in Trizol for RNA extraction.
4.3. Quantitative Real-Time PCR
Trizol (Invitrogen, San Diego, CA, USA) was used to isolate the total mRNA of both tissues and cells. The mRNA samples were reverse transcribed into cDNA using a commercial RT-PCR Kit according to the manufacturer’s instructions (Thermo scientific, Waltham, MA, USA). Relative PCR quantification was performed using a commercial RT-PCR Kit according to the manufacturer’s instructions (TaKaRa, Japan). Expression data were normalized to the amount of Cyclophilin
mRNA using the –ΔΔCt
method. Primers synthesized by AUGCT (Beijing, China) are listed in Table 1
4.4. Western Blot Analysis
Previously described procedures were used [51
]. Briefly, protein samples (20 µg) were separated using 10% SDS-PAGE gels, then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% nonfat dry milk in TBS containing 0.1% Tween for 1 h at room temperature and then blotted with primary antibodies (anti-Adiponectin (1:500) (#2789) (Cell Signaling Technology, Inc., Beverley, MA, USA), anti-β-tubulin (1:1000) (sc-9104) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight. After washing, membranes were incubated with a secondary horseradish peroxidase (HRP)-coupled antibody and visualized using Immobilon HRP substrate (Millipore). The density of the bands was quantified using ImageJ Software (National Institute of Health, Bethesda, MD, USA). The ratio of the intensity of the target protein to that of β-tubulin loading control was calculated to represent the expression level of the protein.
4.5. Plasma Adipokines Measurement
The plasma sample from each mouse was obtained by centrifuging at 900× g in 4 °C for 15 min after collection. The extracted plasma was frozen at −80 °C until analysis. Concentrations of Adiponectin and Leptin in plasma samples were measured using the Mouse Adiponectin and Leptin ELISA kit (Thermo scientific) on an automatic imark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA) according to manufacturer’s procedures.
4.6. Statistical Analysis
Statistical analyses were performed with GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Values were expressed as means ± S.E.M. of independent experiments. Comparisons between the two groups were analyzed by paired Student’s t tests. Comparisons among groups were made by one-way ANOVA test. Differences were considered significant when p < 0.05.